Genetic Manipulation

Anke van Eekelen, PhD

Telethon Institute for Child Health Research

100 Roberts RdSubiaco, WA 6008

9489 7886, ankev@ichr.uwa.edu.au

WHY

would you make a genetically manipulated animal ?* To study the gene identity - gene function relationship

•Manipulation of gene expression by genetic manipulation creates animal models (in vivo models), which reveal a transgenic phenotype of the animal

•This transgenic phenotype is the combination of a set of observed characteristics of the animal resulting from transgenesis: → biochemistry

→ anatomy → physiology → behaviour

Gain of Function-model : Additional copy of a gene - overexpressionAberrant form of a gene - targeted gene mutation

Loss of Function-model : Gene deletion by replacement - knockout animal

These models may reveal the mechanism/pathwayunderlying

a specific outcome or disease

HOW

to make a genetically manipulated animal?

Transgenic mice: pronuclear/oocyte injectionof targeting vector/construct containing the DNA of interest

Knockout mice : blastocyst injectionof transformed embryonic stem cells expressing your gene of interest

Pronuclear injection to make transgenic mouse

Foreign DNA injected is a construct/vector containing:- full coding sequence of the gene of interest- promotor determining tissue specificity & strength

of expression

* Site of integration of injected DNA into genome is random!

* Number of copies of injected DNA into genome is random!single insertion - tandem (>100) array

range of foreign gene expressionrange of phenotypes

• developed transgenics = founders

• Founders are hemizygous fortransgene!

2 cell stage 8 cell stageMORULA

16 cell stageMORULA

BLASTULAIntegration of transgene before first cell division

↓developing embryo will have transgene present inevery somatic & germ line cell(10-30%) Integration of transgene after 2 cell stage

↓Developing embryo = chimera (genetic mosaic)(10%)

Cloning(Ultimate transgenesis)

transfer of a complete “somatic cell nucleus”transfer of a complete “somatic cell nucleus”

www.ozgene.com

blastocystblastocyst

complete adult udder cell fused with an enucleated oocyte by electrical

pulseOocyteOocyte

Sheep “Dolly”Sheep “Dolly”somatic cellsomatic cell

Wilmut, I., et al.,’98

www.ozgene.com

HOW

to make a genetically manipulated animal?

Transgenic mice: pronuclear/oocyte injectionof targeting vector/construct containing the DNA of interest

Knockout mice : blastocyst injectionof transformed embryonic stem cells expressing your gene of interest

Transgenic mouse versus Knockout mouse

- random integration into genome

-Tandem arrays= multiple copies

- fertilized oocyte- site specific integration of transgene

- homologous recombination

replacement vector containing:* two flanking regions of DNA homologous

to the genomic target locus* positive and negative selection markers

- blastocyst

Blastocyst

Inner Cell Mass (ICM) develops into embryo

blastocoel

trophectoderm

www.ozgene.com

Gene-knockout/in procedure in a nutshell

www.ozgene.com

Bacterial gene neo (neomycin phosphotransferase)Causes resistence to drug G418

Tyrosine kinase gene causes sensitivity to drug gancyclovir

Homologous Recombination

1st generation = F1 = founder generation

Blastula injection to makeknockout mousePluripotent murine embryonic

stem cells from blastocyst→

Reporter Mice ?

QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

Okabe M, Okabe M, IkawaIkawa M et al ‘97M et al ‘97

www.ozgene.com

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Transgenic reporter mice-A: randomly integrated reporter geneunder ubiquiteous promoter(green mouse phenotype)

-B: randomly integrated reporter geneunder cell specific promoter (spatial control)

Knockin reporter mice: * Knockout and knockin at same time* Replacement construct contains:

* two flanking regions of DNA homologous to the genomic target locus* positive and negative selection markersBut also….* full coding sequence of another gene than target gene

→ this new gene will be under control of the promotor of the target gene

Knockin reporter mouse

Scl allele:

LacZ KI

1a 1b 2 3 4 5 6 exon

Whole mount LacZ staining LacZ staining on cryosections

Gene deletion in knockout model → mouse phenotype

PhenotypicdifferenceLethal Normal

Targeted gene is eitherunimportant

or redundant

Targeted gene is critical for

development/ survival

Function of targeted gene is revealed

Conditional gene deletion

Conditional gene deletion model

Alternative approach to conventional knockout gene deletion is required:

↓Spatial and/or temporal control over gene deletion

Condition gene deletion models:

* mice express a combination of transgenic and knockin alleles* simultaneous expression of these genetically manipulated alleles

underlying gene deletion*gene deletion is happening in vivo !!

Spatial and/or temporal control over gene deletion

* tissue specific promotorcontrols transgene expression

* developmental stage specific promotorcontrols transgene expression

* inducible activity of expressed transgene

Different conditional gene deletion models: * Cre-loxP / CreER(T)* Tet-On or Tet-Off

Cre-loxP system

Principle: gene deletion by DNA recombination

Cre-loxP system

Cre recombinase LoxP - DNAsequence

* Bacteriophage P1 * Two 13 bp inverted repeats interupted by 8 bp

* mediates site specific nonpalindromic sequence recombination between (34 bp in total length)two lox P sites

TRANSGENE KNOCKIN

ATA ACT TCG TAT AATA ACT TCG TAT A gcgc at ac atat ac at T ATA CGA AGT TATT ATA CGA AGT TAT

Cre-LoxP DNA recombination

crecre++loxP loxP

++loxP

loxP

www.ozgene.com

Knockin mouse= floxed mouse

Transgenic mouse= Cre-mouse

Gene X floxed / Cre+

Genomic sequence for gene X is recombined: Gene X expression

Example: brain specific KO of SCL gene(Cre-loxP; spatial)

Nestin promotor

Conditional SCL-KO in brain

Nestin: * pro-neural gene* specific expression

in brain tissue

Scl: * hematopoietic regulator* expressed in brain tissue* knockout is lethal

Inducible transgenic mouse modelCreER(T)-loxP system

(spatial & temporal)

Tissue specific promoter + Cre ER(T)* Spatial * Temporal

ER(T): mutated estrogen binding domain with affinity only for Tamoxifen (= estrogen antagonist) ↓

Cre ER(T) : * fusion protein behaving like a steroid receptor

* Tamoxifen binding to Cre ER(T) in cytoplasminduces translocation of of cre to nucleus

* In cell nucleus cre can achieve DNA recombination of a floxed gene

Cre ER(T)

Cre ER(T) TAM

Cre-ERTTissue spec. propmoter

lox P sites

Gene X: allele 1

TAM

Inducible Cre-ER(T) / loxP model for gene deletion

Inducible gene deletion models

* Cre-loxP based models

Tamoxifen CreER(T)

Alternative inducers for Cre recombinase:

RU486 CrePRRU486 or Dexamethasone CreGRInterferon Mx1promoter-Cre

* Tet-based models

Tetracycline Tet-On or Tet-Off

In summary:

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Transgenic mouse: random genomic integration of transgene

Knockout/in mouse: site-specific genomic integration of transgene(targeted)

Conditional transgenic: tissue or time specific expression of transgene(random or targeted)

Inducible transgenic: tissue and time specific expression of transgene(random and targeted)

Alternative gene manipulation approaches:

1- RNAi

2- Morpholinos/antisense hybridisation

Alternative approach to make transgenic mice:

1- lentiviral infection (retrovirus, which incorporates in genome)

post transcriptional gene silencing

RNA interference pathway

siRNA

‘Dicer’ ribonuclease

RNA-induced silencing complex

(RISC)

Morpholino Antisense oligos

- bind and inactivate selected RNAs

- fast, simple and most effective for validation of new therapeutic targets(modern drug development)

- potentially effective therapeutics inviral diseases and cancer

-Specific, stable and no non-antisense activity* Morpholino ring with 1 of 4 genetic bases

(replacing the ribose backbone of endogenous RNA)* non-ionic phosphorodiamidate inter-subunit

But … NO GERMLINE TRANSMISSION!!!

Lentiviral genetic manipulation

““TransgenesTransgenes go retro” Nature Biotech Jan. ‘99go retro” Nature Biotech Jan. ‘99

www.ozgene.com

Lentiviraltransgenesisof siRNA

RNA interference pathway

siRNA

‘Dicer’ ribonuclease

RNA-induced silencing complex

(RISC)